Optimizing separations in online comprehensive two‐dimensional liquid chromatography

Online comprehensive two‐dimensional liquid chromatography has become an attractive option for the analysis of complex nonvolatile samples found in various fields (e.g. environmental studies, food, life, and polymer sciences). Two‐dimensional liquid chromatography complements the highly popular hyphenated systems that combine liquid chromatography with mass spectrometry. Two‐dimensional liquid chromatography is also applied to the analysis of samples that are not compatible with mass spectrometry (e.g. high‐molecular‐weight polymers), providing important information on the distribution of the sample components along chemical dimensions (molecular weight, charge, lipophilicity, stereochemistry, etc.). Also, in comparison with conventional one‐dimensional liquid chromatography, two‐dimensional liquid chromatography provides a greater separation power (peak capacity). Because of the additional selectivity and higher peak capacity, the combination of two‐dimensional liquid chromatography with mass spectrometry allows for simpler mixtures of compounds to be introduced in the ion source at any given time, improving quantitative analysis by reducing matrix effects. In this review, we summarize the rationale and principles of two‐dimensional liquid chromatography experiments, describe advantages and disadvantages of combining different selectivities and discuss strategies to improve the quality of two‐dimensional liquid chromatography separations.     

Sequential fractionation procedure for the identification of potentially cytochrome P4501A-inducing compounds

A multistep fractionation procedure for the separation of nonpolar aromatic compounds with respect to cytochrome P4501A induction is presented. Normal-phase HPLC on nitrophenylpropyl silica and cyanopropyl silica was tested for group-specific separation as a first fractionation step. Subsequent individual compound-specific PAH fractionation was done by means of reversed-phase HPLC. Electron-donor-acceptor HPLC and size-exclusion chromatography were applied to separate PAHs, PCBs, PCNs and PCDD/Fs according to their number of aromatic carbon atoms, their hydrophobicity, their degree of chlorination, their planarity and their molecular size. The method was validated for complex environmental mixtures on the basis of two sediment extracts.     

Comprehensive two-dimensional high performance liquid chromatography system with immobilized liposome chromatography column and monolithic column for separation of the traditional Chinese medicine Schisandra chinensis

A comprehensive two-dimensional (2D) separation is one that employs two separation dimensions (columns) and draws on all of the available resolving power from each of the dimensions of separate the components in a sample. In this study, a comprehensive 2D chromatography approach was developed for the separation and identification of membrane permeable compounds in a famous traditional Chinese medicine of Schisandra chinensis. The first dimensional column was the immobilized liposome chromatography (ILC) column, which mimics the biological membranes and can be used to study drug-membrane interactions in liquid chromatography. Using an automatic ten-port switching valve equipped with two sample loops, the section of the first-dimension was introduced in the second-dimension consist of a silica monolithic column. More than 40 components in Schisandra chinensis were resolved by using the developed separation system and among them 14 compounds were identified interacting with the ILC column based on their retention action, UV and mass data. With this comprehensive 2D-HPLC system, the three-dimensional chromatographic fingerprints of Schisandra chinensis were preliminarily established and processed by using principal component analysis and hierarchical clustering analysis. The obtained information can distinguish the unacceptable samples of the quality control. The result demonstrated that the 2D biochromatography system has been demonstrated to have more advantages of finding strong binding bioactive components, providing an enhanced peak capacity, good sensitivity and powerful resolution biological fingerprinting analysis of complex TCMs, which was a useful means to control the quality of and to clarify the membrane permeability of the compounds in Schisandra chinensis.     

Comprehensive two‐dimensional monolithic liquid chromatography of polar compounds

Two‐dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On‐line coupling of a reversed‐phase column with an aqueous normal‐phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on‐line two‐dimensional liquid chromatography needs a capillary or micro‐bore column providing low‐volume effluent fractions transferred to a short efficient second‐dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro‐columns in fused silica capillaries with structurally different dimethacrylate cross‐linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed‐phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on‐line an organic polymer monolithic capillary column in the first dimension with a short silica‐based monolithic column in the second dimension provides two‐dimensional liquid chromatography systems with high peak capacities. The silica monolithic C₁₈ columns provide higher separation efficiency than the particle‐packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed‐phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.     

CE separation of various analytes of biological origin using polyether ether ketone capillaries and contactless conductivity detection

The development of efficient and sensitive analytical methods for the separation, identification and quantification of complex biological samples is continuously a topic of high interest in biological science. In the present study, the possibility of using a polyether ether ketone (PEEK) capillary for the CE separation of peptides, proteins and other biological samples was examined. The performance of the tubing was compared with that of traditional silica capillaries. The CE analysis was performed using contactless conductivity detection (C⁴D), which eliminated any need for the detection window and was suitable for the detection of optically inactive compounds. In the PEEK capillary the cathodic EOF was low and of excellent stability even at extremes pH. In view of this fast biological anions were analyzed using an opposite end injection technique without compromising separation. A comparison of the performances of fused‐silica and polymer capillaries during the separation of model sample mixtures demonstrated the efficiency and separation resolution of the latter to be higher and the reproducibility of the migration times and peak areas is better. Furthermore, PEEK capillaries allowed using simple experimental conditions without any complicated modification of the capillary surface or use of an intricate buffer composition. The PEEK capillaries are considered as an attractive alternative to the traditional fused‐silica capillaries and may be used for the analysis of complex biological mixtures as well as for developing portable devices.     

Separation of alkylnaphthalenes by preparative liquid chromatography using column switching systems

Summary The paper describes the separation of the mixture of alkynaphthalenes from distillation cuts of a pyrolysis oil, by preparative liquid chromatography on silica. The design of the system permits the connection of the columns to form multicolumn systems.The samples were first separated on a single column. The mixtures were further separated using two-column chromatography systems.The obtained fractions were analyzed by capillary gas chromatography. In most cases a substantial increase in the concentrations of the individual components was achieved. In several cases, pure compounds have been obtained. Separation efficiency increases in the following order: single column, two directly coupled collumns, two-step switching chromatography, heartcutting.     

Comprehensive two-dimensional gas chromatography: a hyphenated method with strong coupling between the two dimensions.

Comprehensive two-dimensional gas chromatography (GC x GC) provides a true orthogonal separation system. It is explained and demonstrated that it generates a peak capacity that is approximately equal to the product of the peak capacities of the two individual separation systems. The resulting peaks are ordered in a two-dimensional plane in bands of compounds with the same characteristics. Quantitation of the separated (groups of) components is fundamentally not different from one-dimensional gas chromatography, but the sensitivity is far better and true baseline is always available. The two co-ordinates of each peak in the plane make the identification more reliable. Instrumental considerations of GC x GC are discussed. The three designs of contemporary GC x GC systems are presented and compared. Although the technique is still very young, a number of applications on complex samples as petroleum and environmental samples have already been reported. Finally, the future perspectives of GC x GC are discussed.     

Comparison of Mobile Phases for Separation and Quantification of Lipids by One-Dimensional TLC on Preadsorbent High Performance Silica Gel Plates

Abstract

Twenty-four solvent systems reported in the literature for the one-dimensional TLC separation of lipids and phospholipids were compared under identical conditions using high performance preadsorbent silica gel plates. The best overall separation of mixtures of neutral lipid and phospholipid standards and compounds extracted from the digestive gland-gonad complex of Biomphalaria glabrata snails was obtained with a system utilizing consecutive development with chloroform-methanol-water (65:25:4), chloroform-hexane (3:1), and carbon tetrachloride. The best system for quantification of neutral lipids was hexane-diethyl ether-formic acid (80:20:2). R_f data are tabulated and results discussed for all systems tested.     

Separation of coumarins from Archangelica officinalis in high-performance liquid chromatography and thin-layer chromatography systems

Complex, multicomponent mixtures are difficult to separate in a single chromatographic run. Therefore, the possibility to separate twelve coumarins from Archangelica officinalis was studied by combining a HPLC and a TLC system. HPLC optimized by the use of DryLab for Windows software was performed on RP-18 column and TLC was performed on silica plates. Fractions from the RP column were evaporated, applied on silica plate and developed in non-aqueous solvent. Possibilities of complete separation of investigated coumarins were discussed in RP and NP systems. The result of their complete separation was presented by HPLC chromatograms, DryLab simulated chromatograms and a video scan of TLC plate.     

The generally useful estimate of solvent systems method facilitates off-line two-dimensional countercurrent chromatography for isolating compositions from Siraitia grosvenorii roots

Solvent system selection is a crucial and the most time-consuming step for successful countercurrent chromatography separation. A thin-layer chromatography-based generally useful estimate of solvent systems method has been developed to simplify the solvent system selection. We herein utilized the method to select a solvent system for off-line two-dimensional countercurrent chromatography to separate chemical compositions from a complex fraction of the Siraitia grosvenorii root extract. The first-dimensional countercurrent separation using chloroform/methanol/water (10:5.5:4.5, v/v/v) yielded four compounds with high purity and three mixture fractions (Fr I, III, and VII). The second-dimensional countercurrent separation conducted on Fr I, III, and VII using the hexane/ethyl acetate/methanol/water (4:6:6:4, 3:7:3:7, v/v/v) and chloroform/methanol/water (10:9:6, v/v/v) solvent systems, respectively, produced another four compounds. Four triterpenoids and four lignans were finally isolated, including two novel compounds. Hence, the generally useful estimate of solvent systems method is a feasible and efficient approach for selecting an applicable solvent system for separating complex samples. In addition, the off-line two-dimensional countercurrent chromatography method can improve both the peak resolution and the capacity of countercurrent chromatography.     

Reversed-phase systems for the separation of pentapeptides by high-performance liquid chromatography

Summary Reversed-phase systems using octyl modified silica as such and as a support for dynamically coated ion-exchangers, were investigated for their ability to separate pentapeptides. Normal reversed-phase adsorption with C-8 bonded silica in combination with citrate bufferpropanol-1 mixtures were found useful for the separation of a number of pentapeptides. The separation of pentapeptides differing widely in retention can be speeded up by applying an organic modifier and/or sodium citrate gradient. A solvent generated cation-exchange system with sodium dodecylsulfate as surfactant showed a high selectivity for the pentapeptides under investigation and is better for analytical purposes than the normal reversed-phase adsorption systems investigated. With respect to the detection of pentapeptides with fluorescamine, the use of dry pyridine as a basic buffer and as diluent for the fluorescamine was also investigated. Compared to the commonly used diluent acetone, pyridine is better when using acidic eluents of moderate buffer strength. At pH>6 no significant differences in sensitivity between acetone and pyridine could be noticed.     

Preconcentration of petroleum organic acids and ketones by two-stage chromatography using a modified adsorbent

A procedure was developed for preconcentrating organic acids and ketones present in oils from Western Siberia, petroleum products, and hydrocarbon mixtures. The procedure is based on two-stage chromatography in a system containing a precolumn with silica gel modified with 10% potassium silicate and a separation column packed with untreated silica gel. Elution was performed using solvents with an increasing polarity. The efficiency of isolation was tested using standard compounds.     

Validation of theory of n-column separations with gas chromatograms predicted by commercial software

A probability theory for the average number of compounds resolved by the partial separation of complex mixtures on n columns was tested using commercial-software predictions of gas chromatograms. Such n-column separations are traditional means for addressing peak overlap, in which one chooses additional columns of different selectivity to separate compounds that cannot be separated by a single column. Gas chromatograms of five types of complex mixtures containing from 99 to 283 compounds were predicted for eight stationary phases using both optimized and other temperature programs. The number n of columns for different mixtures varied from 2 to 5. The numbers of compounds separated as singlet peaks at different resolution thresholds were compared to predictions, as evaluated with point-process statistical-overlap theory based on a Poisson distribution. A good agreement between theory and results was found in all cases corresponding to low saturation. Both good and poor agreements were found for cases corresponding to high saturation. A good agreement also was found for results based on resolving complex mixtures by a single column subject to two temperature programs. The moments and distribution of the number of resolved compounds were computed by Monte Carlo simulation, thus gauging the significance of departures between results and theory. The potential of such simulations to explore the limitations of theory was briefly investigated.     

High‐performance liquid chromatography with electrospray ionization ion mobility spectrometry: Characterization,data management,and applications

The combination of high‐performance liquid chromatography and electrospray ionization ion mobility spectrometry facilitates the two‐dimensional separation of complex mixtures in the retention and drift time plane. The ion mobility spectrometer presented here was optimized for flow rates customarily used in high‐performance liquid chromatography between 100 and 1500 μL/min. The characterization of the system with respect to such parameters as the peak capacity of each time dimension and of the 2D spectrum was carried out based on a separation of a pesticide mixture containing 24 substances. While the total ion current chromatogram is coarsely resolved, exhibiting coelutions for a number of compounds, all substances can be separately detected in the 2D plane due to the orthogonality of the separations in retention and drift dimensions. Another major advantage of the ion mobility detector is the identification of substances based on their characteristic mobilities. Electrospray ionization allows the detection of substances lacking a chromophore. As an example, the separation of a mixture of 18 amino acids is presented. A software built upon the free mass spectrometry package OpenMS was developed for processing the extensive 2D data. The different processing steps are implemented as separate modules which can be arranged in a graphic workflow facilitating automated processing of data.     

Separation of isotopic compounds by reversed-phase liquid chromatography. Effect of pressure gradient on isotope separation by ionization control

Isotope separation utilizing ionization control in reversed-phase liquid chromatography was studied. Several organic acids and bases labeled with oxygen-18 and nitrogen-15 were successfully separated from unlabeled compounds by using either 6-m microbore columns or recycle chromatography with short columns of conventional size. Differences in the separation factors for the same isotopic mixtures were observed between the two chromatographic systems, and attributed to the difference in the pressure gradient along the columns in the two systems. The steep pressure gradient existing in the short recycle columns resulted in an increase in the peak width and the separation factor for oxygen isotopic compounds, and a decrease in the peak width and the separation factor for nitrogen isotopic compounds.     

Electrophoretic separations in poly(dimethylsiloxane) microchips using mixtures of ionic,nonionic and zwitterionic surfactants

The use of surfactant mixtures to affect both EOF and separation selectivity in electrophoresis with PDMS substrates is reported, and capacitively coupled contactless conductivity detection is introduced for EOF measurement on PDMS microchips. First, the EOF was measured for two nonionic surfactants (Tween 20 and Triton X‐100), mixed ionic/nonionic surfactant systems (SDS/Tween 20 and SDS/Triton X‐100), and finally for the first time, mixed zwitterionic/nonionic surfactant systems (TDAPS/Tween 20 and TDAPS/Triton X‐100). EOF for the nonionic surfactants decreased with increasing surfactant concentration. The addition of SDS or TDAPS to a nonionic surfactant increased EOF. After establishing the EOF behavior, the separation of model catecholamines was explored to show the impact on separations. Similar analyte resolution with greater peak heights was achieved with mixed surfactant systems containing Tween 20 and TDAPS relative to the single surfactant system. Finally, the detection of catecholamine release from PC12 cells by stimulation with 80 mM K⁺ was performed to demonstrate the usefulness of mixed surfactant systems to provide resolution of biological compounds in complex samples.     

Effect of chromatographic conditions on the separation and system efficiency for HPLC of selected alkaloids on different stationary phases

Retention parameters of alkaloid standards were determined on different stationary phases, i.e., octadecyl silica, base-deactivated octadecyl silica, cyanopropyl silica, preconditioned cyanopropyl silica, and pentafluorophenyl, using different aqueous eluant systems: acetonitrile-water mixtures; buffered aqueous mobile phases at pH 3 or 7.8; and aqueous eluants containing ion-pairing reagents (octane-1-sulfonic acid sodium salt and pentane-1-sulfonic acid sodium salt) or silanol blockers (tetrabutyl ammonium chloride and diethylamine). Improved peak symmetry and separation selectivity for basic solutes was observed when basic buffer, ion-pairing reagents, and, especially, silanol blockers as mobile phase additives were applied. The best separation selectivity and most symmetric peaks for the investigated alkaloids were obtained in systems containing diethylamine in the mobile phase. The influence of acetonitrile concentration and kind and concentration of ion-pairing reagents or silanol blockers on retention, peak symmetry, and system efficiency was also examined. The most efficient and selective systems were used for separation of the investigated alkaloids and analysis of Fumaria officinalis and Glaucium flavum plant extracts.     

亲水/反相二维制备液相色谱法分离纯化络石藤中的化学成分

建立了亲水/反相二维制备液相色谱(Pre-2D-HILIC/RPLC)分离纯化络石藤中化学成分的分析方法。络石藤药材经醇提、活性炭脱色后用反相固相萃取柱除去色素和强极性物质,最终得到干燥的浅黄色粉末。一维亲水色谱选择Click XIon色谱柱(250 mm×20 mm,10μm)作为固定相,水和乙腈作为流动相,进行梯度洗脱,以紫外触发模式收集馏分,共得到15个组分。二维反相色谱选择C18色谱柱(250 mm×20 mm,5μm)作为固定相,水和乙腈作为流动相,进行梯度洗脱,最终得到14个高纯度化合物,并通过质谱和核磁共振对其进行确认。实验结果表明,该法具有良好的正交选择性,可以有效提高分离度和峰容量,对于分离络石藤等复杂样品具有重要意义。     

Optimization by orthogonal array design of ion-pair HPLC separation of the enzymatic hydrolysis products of yeast RNA

<正>The separation of enzymatic hydrolysis products of yeast RNA by ion-pair HPLC was studied.A modified chromatographic response function(MCRF) was proposed to appraise the effectiveness of chromatographic separation.This function takes the number of peaks,resolution and the retention time of the last peak into consideration.It shows advantages for optimization of HPLC separation of complex mixtures.An orthogonal array design was used to separate the hydrolysate of yeast RNA and the optimal chromatographic conditions were obtained.     

A New Dimension in Separation Science: Comprehensive Two‐Dimensional Gas Chromatography

The introduction and development of comprehensive two‐dimensional gas chromatography offers greatly enhanced resolution and identification of organic analytes in complex mixtures compared to any one‐dimensional separation technique. Initially promoted by the need to resolve highly complex petroleum samples, the technique’s enormous separation power and enhanced ability to gather information has rapidly attracted the attention of analysts from all scientific fields. In this Minireview, we highlight the fundamental theory, recent advances, and future trends in the instrumentation and application of comprehensive two‐dimensional column separation.